Method for inducing hepatocyte proliferation and uses thereof

ABSTRACT

The present application provides methods and compositions for inducing hepatocyte proliferation and liver regeneration, the latter being mainly dependent on hepatocyte proliferation even if all the other cell types divide to reconstitute the organ specific-lobular-architecture. The methods and compositions provided herein make use of an A 3 AR agonist. A preferred A 3 AR agonist disclosed herein is Cl-IB-MECA.

FIELD OF THE INVENTION

This invention relates to the field of therapeutics and in particular tomethods for inducing hepatocyte differentiation and liver regeneration.

PRIOR ART

The following is a list of art which is considered to be pertinent fordescribing the state of the art in the field of the invention.Acknowledgement of these references herein will at times be made byindicating their number within brackets from the list below.

-   1. Henrion J. Ischemia/reperfusion injury of the liver:    pathophysioloic hypotheses and potential relevance to human hypoxic    hepatitis. Acta Gastroenterol Belg. 63:336-347.-   2. Shirasugi N, Wakabayashi G, Shimazu M. Up-regulation of    oxygen-derived free radicals by interleukin-1 in hepatic    ischemia/reperfusion injury. Transplantation 64:1398-403.-   3. Xu Z, Jong Y, Mueller R A, Norfleet EA: IB-MECA and    cardioprotection. Cardiovasc. Drug Rev. 24(3-4):227-238.-   4. Chen G J, Harvey B K, Shen H, Chou J, Victor A, Wang Y:    Activation of adenosine A3 receptors reduces ischemic brain injury    in rodents. J. Neurosci. Res. 84(8):1848-1855.-   5. Fishman P, Bar-Yehuda S, Farbstein T, Barer F, Ohana G: Adenosine    acts as a chemoprotective agent by stimulating G-CSF production: a    role for A1 and A3 adenosine receptors. J. Cell. Physiol.    183(3):393-398.-   6. Fishman P, Bar-Yehuda S, Barer F, Madi L, Multani A F, Pathak S:    The A3 adenosine receptor as a new target for cancer therapy and    chemoprotection. Exp. Cell. Res. 269(2): 230-236.-   7. Bar-Yehuda S, Madi L, Barak D et al.: Agonists to the A3    adenosine receptor induce G-CSF production via NF-kappaB activation:    a new class of myeloprotective agents. Exp. Hematol.    30(12):1390-1398.-   8. Fishman P, Bar-Yehuda S, and Wagman L. (1998). Adenosine and    other low molecular weight factors released by muscle cells inhibit    tumor cell growth: Possible explanation for the rarity of metastases    in muscle. Cancer Res. 58:3181-3187.-   9. Fishman P, Bar-Yehuda S, Farbstein T, Barer F, and Ohana G.    Adenosine acts as a chemoprotective agent by stimulating G-CSF    production: A role for A1& A3 adenosine receptors. J. Cell. Physiol.    183:393-398.-   10. Fishman P, Bar-Yehuda S, Barer F, Madi L, Multani Asha S,    Pathak S. The A3 adenosine receptor as a new target for cancer    therapy and chemoprotection. Exp Cell Res. 269:230-236.-   11. Teoh N, dela Pena A, Farrell G. Hepatic ischemia preconditioning    in mice is associated with activation of NF_(K)B. p38 kinase and    cell cycle entry. Hepatology 36:94-102.

BACKGROUND OF THE INVENTION

The liver is the only vital organ, aside from the brain, for which thereis no pharmacological, mechanical, or extra corporeal means of supportfor a failing organ, such as are found for the lungs, kidney and heart.The liver is also unique in that it is the only mammalian organ that canregenerate its biologically functional parenchymal mass followingresection or injury, instead of healing with biologically nonfunctionalscar tissue.

Liver resections have become safer in the past 10 years, owing toimprovements in preoperative diagnosis, surgical techniques andpostoperative care. Postoperative mortality correlates directly withpreoperative liver function and resected liver volume. Function of theremnant liver rapidly recovers in patients with normal liver parenchymaas hepatocytes proliferate to restore the loss of volume. However, inthe presence of parenchymal liver disease, as in patients with livercirrhosis, severe liver steatosis or colorectal liver metastasis,debilitated by neoadjuvant chemotherapy before liver resection,hepatocellular proliferation is impaired, exposing patients to liverdysfunction and associated complications, culminating in posthepatectomyliver failure, which has a high mortality (60-90%).

Several pathways have been identified in the regenerating liverincluding a cytokine pathway that is largely responsible for the entryof hepatocytes into the cell cycle (transition from G0 to G1), a processthat is known as priming, and a growth factor pathway that isresponsible for cell-cycle progression (G1 phase to the S phase).

In addition, ischemia-reperfusion injury of the liver is another known,clinically significant manifestation of surgical procedures, such asliver transplantation and partial hepatic resection (1). There are twodistinct phases of liver injury after ischemia-reperfusion injury. Theinitial phase (<2 h after reperfusion) is characterized by oxidantstress, where production and release of reactive oxygen species (ROS)appears to directly result in hepatocelluar injury. The late phase (6-48h after reperfusion) is an inflammatory disorder mediated by recruitedneutrophils. Interrelationships between products of activated Kupffercells and neutrophils, such as tumor necrosis factor (TNF-α),interleukin (IL)-1, nitric oxide (NO) and leukotrienes, have beenimplicated in the pathogenesis of hepatic ischemia-reperfusion injury(2). The biological effects of TNF-α extend from inducing cell death topromoting cell regeneration.

Indeed, recent studies have shown that ischemic preconditioning may beassociated with entry of hepatocytes into the cell cycle within 2 h ofsubsequent ischemia-reperfusion in a murine model of partial hepatic IRinjury (11).

Adenosine, through its binding to selective G-protein-associatedmembrane receptors, designated as A₁, A_(2A), A_(2B) and A₃, accumulatesextracellularly following ischemia, and is known to confercytoprotection. In particular, the A₃AR has been found to be involved inmediating cardio-neuro- and chemo-protection (3-7).

The A₃ adenosine receptor, a G_(i) protein-associated cell surfacereceptor, has been proposed as a target to combat cancer andinflammation. The receptor is highly expressed in various tumor celltypes while low expression was shown in adjacent normal tissues. In vivostudies have shown that A₃AR agonists inhibit the development of colon,prostate and pancreatic carcinomas as well as melanoma and hepatoma.

A₃AR agonists were also been shown to act as anti-inflammatory agents byameliorating the inflammatory process in different experimentalautoimmune models such as rheumatoid arthritis, Multiple sclerosis andCrohn's disease.

Moreover, A₃AR agonists have been shown to possess a differential effecton tumor and normal cell growth. While activation of the A₃AR inhibitsthe growth of various tumor cell lines, it stimulates the proliferationof normal cells such as bone marrow cells (8-10).

At present, there is no pharmacological intervention proven to eitherattenuate liver cell injury or to augment tissue regeneration of theliver after acute or chronic injury of this vital organ.

SUMMARY OF THE INVENTION

The present invention is based on the following findings obtained fromexperiments conducted in rats with Cl-IB-MECA, a specific and selectiveA₃AR agonists:

-   -   treatment with Cl-IB-MECA induced proliferation of hepatocytes        after partial hepatectomy;    -   treatment with Cl-IB-MECA reduced serum levels of the liver        enzymes alanine aminotransferase (ALT) and aspartate        aminotransferase (AST) after partial hepatectomy;    -   treatment with Cl-IB-MECA increased the number of hepatocytes        undergoing mitosis upon partial hepatectomy;    -   treatment with Cl-IB-MECA increased the number of proliferation        cell nuclear antigen (PCNA) positive cells (the expression        correlating with degree of cell proliferation);    -   treatment with Cl-IB-MECA increased liver weight.

Based on the above findings it has been concluded that A₃AR agonists maybe utilized for inducing hepatocytes' proliferation and liverregeneration.

Thus, in accordance with a first aspect, there is provided a method ofstimulating hepatocytes' proliferation, comprising contactinghepatocytes with an A₃AR agonist in an amount effective to stimulatehepatocytes' proliferation.

Further provided by the present disclosure is an A₃AR agonist for use ina method for stimulating hepatocytes' proliferation.

In another embodiment, there is provided by the present disclosure anA₃AR agonist for use in a method for treating a patient having liverdamage, e.g. caused by a disease, e.g. cirrhosis, caused by a surgicaltreatment, e.g. due to hepatectomy and others.

The present disclosure also provides the use of an A₃AR agonist for thepreparation of a medicament for treating a liver damage in a patient.

Further, there is provided by the present disclosure a pharmaceuticalcomposition for stimulating hepatocyte proliferation, comprising an A₃ARagonist in an amount effective to stimulate hepatocytes' proliferation.Also provided by the present disclosure is a pharmaceutical compositionfor treating a liver damage, comprising an A₃AR agonist in an amounteffective to stimulate hepatocytes' proliferation.

In one embodiment, the A₃AR agonist is2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methyl-uronamide (Cl-IB-MECA),and in another embodiment, the A₃AR agonist isN⁶-(3-iodobenzyl)-adenosine-5′-N-methyl-uronamide (IB-MECA). However,these currently preferred A₃AR agonists are by no means exclusive andother such A₃AR agonists may also be used, as detailed further below.

In the context of the present disclosure, a variety of conditions inwhich the liver or liver cells are damaged following liver injury,hepatectomy, disease-induced or infection-induced liver damage may betreated by the use of an A₃AR agonist.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carriedout in practice, embodiments will now be described, by way ofnon-limiting example only, with reference to the accompanying drawings,in which:

FIG. 1 is a bar graph showing the effect of Cl-IB-MECA (Treated) on theproliferation of hepatocytes in the liver of a rat undergoing liverregeneration after partial hepatectomy;

FIGS. 2A and 2B are bar graphs showing the serum levels of the liverenzymes alanine aminotransferase (ALT) (FIG. 2A) and aspartateaminotransferase (AST) (FIG. 2B) in rats treated with Cl-IB-MECA ascompared to control rats, 2, 4 and 48 hours after partial hepatectomy.

FIG. 3 is a bar graph showing serum levels of the liver enzymes AST andALT in rats which underwent partial hepatectomy and treated Cl-IB-MECAas compared to untreated rats (naïve) or rats treated with vehicle(vehicle).

FIG. 4 is a bar graph showing mitotic index of hepatocytes in rats whichunderwent partial hepatectomy and treated with Cl-IB-MECA, as comparedto rats treated with vehicle (vehicle).

FIGS. 5A-5B are microscopic images following proliferating cell nuclearantigen (PCNA) staining of hepatocytes 48 hours after partialhepatectomy following treatment with vehicle only (FIG. 5A, vehicle) orwith Cl-IB-MECA (FIG. 5B).

FIG. 6 is a bar graph showing liver weight (% of regeneration) 24 and 48hours following partial hepatectomy in Cl-IB-MECA treated or ratstreated with vehicle only (vehicle).

DETAILED DESCRIPTION OF THE INVENTION

The invention is described in the following detailed description withreference to methods for the stimulation of hepatocyte proliferation andliver regeneration. It should be noted that in addition to said methods,also encompassed within the present invention is an A₃AR agonist for usein a method for stimulating hepatocyte proliferation and liverregeneration; the use of an A₃AR agonist for the preparation of apharmaceutical composition for administration to a subject requiringstimulation of hepatocyte proliferation and liver regeneration; as wellas a pharmaceutical composition for the stimulation of hepatocyteproliferation and liver regeneration, that comprises an effective amountof an A₃AR agonist and a pharmaceutically acceptable carrier.

As used in the specification and claims, the forms “a”, “an” and “the”include singular as well as plural references unless the context clearlydictates otherwise. For example, the term “an A₃AR agonist” includes oneor more agonists.

Further, as used herein, the term “comprising” is intended to mean thatthe methods or composition includes the recited elements, but notexcluding others. Similarly, “consisting essentially of” is used todefine methods and compositions that include the recited elements butexclude other elements that may have an essential significance on thestimulation of hepatocyte proliferation and liver regeneration. Forexample, a composition consisting essentially of an A₃AR agonist willnot include or include only insignificant amounts (amounts that willhave an insignificant effect on the anti-inflammatory effect of thecomposition) of other active ingredients that have an hepatocyteproliferation and liver regeneration activity. Also, a compositionconsisting essentially of the active agents as defined herein would notexclude trace contaminants from the isolation and purification method,pharmaceutically acceptable carriers, excipients, preservatives, and thelike. “Consisting of” shall mean excluding more than trace elements ofother elements. Embodiments defined by each of these transition termsare within the scope of this invention.

Further, all numerical values, e.g., concentration or dose or rangesthereof, are approximations which are varied (+) or (−) by up to 20%, attimes by up to 10% of from the stated values. It is to be understood,even if not always explicitly stated that all numerical designationsshould be read as if preceded by the term “about”. It also is to beunderstood, although not always explicitly stated, that the reagentsdescribed herein are merely exemplary and that equivalents of such areknown in the art.

As detailed in the following exemplary embodiment, the invention isbased on the finding that A₃AR agonists may be used to enhance liverregeneration.

Thus, A₃AR agonists may be used in in vitro and in vivo methods ofstimulating hepatocytes and promoting hepatocyte proliferation. Inaccordance with the methods disclosed herein, hepatocytes may becontacted with an amount of an A₃AR agonist effective in inducingproliferation of the hepatocytes.

Such methods and uses include, by one embodiment, the addition of A₃ARagonists to hepatocytes in vitro. Accordingly, there are disclosedmethods and uses in culturing hepatocytes in vitro, e.g. for subsequenttransplantation, for generating artificial liver tissue ex vivo, etc.Such methods and uses involve the provision of a biologically effectiveamount of an A₃AR agonist to an in vitro or ex vivo biological samplethat contains a population of hepatocytes.

Preferred methods, A₃AR agonist, uses and pharmaceutical compositions inthe context of the present disclosure are those in which the A₃ARagonists are intended for induction of proliferation of hepatocytes invivo, within the framework of a therapeutic treatment intended forinducing proliferation of hepatocytes to counter liver damages of thekind noted above and further below. Thus provides herein are methods,A₃AR agonist, and uses of inducing liver growth, stimulating hepaticregeneration and, generally, treating subjects having various forms ofliver damage and disease.

Within the framework of the present disclosure, the term “liver damage”is used to denote any type of hepatic trauma (injury), including chronicand acute trauma as well as pathological change present in liver cell ortissue. The clinical conditions of liver damage may include, withoutbeing limited thereto, degeneration of live cells, vasculitis of liver,spotty necrosis or focal necrosis present in liver, inflammatory cellinfiltration or fibroblast proliferation in liver and portal area, orhepatomegaly, and hepatocirrhosis, hepatoma resulted from severe liverdamage, and the like. The damage may be a result of a disease (i.e.disease induced) and/or toxicity hepatotoxic chemical substance-inducedliver damage. It is known that some drugs can cause liver damage, andresult in hepatic cytolysis and necrosis.

Within the framework of the present disclosure, the A₃AR agonist isadministered to a subject in amounts effective to promote hepatocyteproliferation, induce liver growth, stimulate hepatic regenerationand/or to generally treat or prevent liver damage, diseases and/ordisorders in the animal or human patient. The team “effective amounts”or “amount effective to”, as used in the present specification refers toamounts effective to promote hepatocyte proliferation, induce livergrowth, stimulate hepatic regeneration and/or treat or prevent liverdamage when administered to an animal or human patient. The effectiveamount is preferably an amount yielding a concentration of the A₃ARagonist in which it selectively activates the A₃AR without activatingany other adenosine receptor. For example, in the case of IB-MECA andCl-IB-MECA such a preferred amount is an amount that will yield aconcentration of less than about 200, 150, 125 or even less than about100 nM in the case of IB-MECA and less than about 400, 300, 250, or evenless than about 200 nM in the case of Cl-IB-MECA. The resultingconcentration in an in vitro embodiment where induction of proliferationof hepatocytes is carried out in can be simply calculated or determinedanalytically. In the case of an in vivo administration to achieveproliferation of hepatocytes in vivo, e.g. within the framework oftreating a liver injury, the effective amount may be determined throughpharmacokinetic (PK) studies by measuring blood or plasma concentrationsof the A₃AR agonists at defined time intervals (by blood withdrawal atsuch times) following administration of the A₃AR agonists. In PK studiesthe maximal concentration of the A₃AR agonist in the blood or plasmashould preferably be a concentration which is below that in whichanother adenosine receptor will be activated.

Induction of hepatocyte proliferation in the context of the presentdisclosure denotes the promotion or stimulation of hepatocyte division,and at times, the inhibition of hepatocyte death.

The A₃AR agonists is preferably formulated for systemic administration,including oral, transdermal, intravenous, intraperitoneal, subcutaneousor intramuscular administration. More localized delivery to the liver isalso contemplated, including all forms of intra-hepatic administration.

A wide range of diseases, disorders and conditions associated with liverdamage may be treated by the A₃AR agonists as disclosed herein. Theseinclude liver damage associated with exposure to alcohol, hepatotoxicdrugs and combinations thereof. Exemplary damaging agents areanticonvulsants, phenyloin, carbamazepine and phenobarbital, andrecreations drugs, such as that know as “Ecstasy”(3,4-methylenedioxymethamphetamine).

Side effects resulting from other therapies may also be treated inaccordance with the present disclosure, including the liver damageassociated with exposure to anti-tuberculosis agents andchemotherapeutic agents. The analgetic acetaminophen (i.e., Panadol, thechemical name of which is 4-(N-acetylamino)phenol), when administratedin a large dose, is a kind of liver-damaging substance that can inducenecrosis of human liver. For example, long-term administration ofantibiotic, such as rifampicin, pyrazinamide, and isoniazide, andlong-term administration of estrogen and the like in the period ofmenopause, also can cause severe hepatocyte necrosis, leading to liverdamage, such as acute or chronic hepatitis, jaundice, and hepaticfibrosis and the like.

Liver damage associated with a reduction in viable liver tissue may alsobe treated, such as occurs after resecting a carcinoma.

Liver damage resulting from or associated with infectious agents mayalso be counteracted using the present invention. This includes liverdamage associated with bacterial, parasitic, fungal and viralinfections. For example, liver damage results from Aspergillus fungalinfections, Schistosoma parasitic infections and a variety of viralinfections, such as adenovirus, retrovirus, adeno-associated virus(AAV), hepatitis virus A, hepatitis virus B, hepatitis virus C,hepatitis virus E, herpes simplex virus (HSV), Epstein-Barr virus (EBV)and paramyxovirus infections, all of which may be treated hereby.

In the context of the present disclosure, A₃AR agonists may also beutilized in the treatment or even prevention of liver damage associatedwith excess acetaminophen (paracetamol) ingestion. This may occur over aprolonged time period, leading to chronic liver damage; or during ashort or immediate time period, leading to acute liver damage. Thelatter embodiments include deliberate and accidental overdoses,including in both adults and children.

Various A₃AR agonists are known in the art. However, the invention isnot limited to known A₃AR agonists. Generally, the A₃AR agonist is anycompound that is capable of specifically binding to the adenosine A₃receptor (“A₃AR”), thereby fully or partially activating said receptorto yield a therapeutic effect (in this particular case, an inductiveeffect on hepatocyte proliferation).

The A₃AR agonist is thus a compound that exerts its prime effect throughthe binding and activation of the A₃AR. In accordance with oneembodiment, this would mean that at the doses it is being administeredit essentially binds to and activates only the A₃R. In a preferredembodiment, the A₃AR agonist has a binding affinity (K_(i)) to the humanA₃AR of less than 1000 nM, desirably less than 500 nM, advantageouslyless 200 nM and even less than 100 nM, typically less than 50 nM,preferably less than 20 nM, more preferably less than 10 nM and ideallyless than 5 nM. The lower the K_(i), the lower the dose of the A₃ARagonist (that may be used) that will be effective in activating the A₃Rand thus achieving a therapeutic effect.

By way of example, the IC₅₀ and K_(i) of IB-MECA and Cl-IB-MECA, bothspecific A₃AR agonists are shown in the following Tables 1 and 2:

TABLE 1 Binding Affinities of IB-MECA to the 4 adenosine receptorsReceptor IC₅₀ (nM) K_(i) (nM) A₁ >1,000 Not determined A_(2A) 685 560A_(2B) 47,600 42,300 A₃ 0.68 0.47

TABLE 2 Binding Affinities of Cl-IB-MECA to the 4 adenosine receptorsReceptor IC₅₀ (nM) K_(i) (nM) A₁ 5,390 3,140 A_(2A) 2,090 1,170 A_(2B)No activity No activity A₃ 0.717 0.661

As these tables clearly show, both IB-MECA and Cl-IB-MECA are highlyselective agonists to the A₃AR.

It should be noted that some A₃AR agonists can also interact with andactivate other receptors with lower affinities (namely a higher K_(i)).A compound will be considered an A₃AR agonists in the context of thepresent disclosure (namely a compound that exerts its prime effectthrough the binding and activation A₃AR) if its affinity to the A₃AR isat least 3 times (i.e. its K_(i) to the A₃AR is at least 3 times lower).Preferably the A₃AR agonist used in the context of the presentdisclosure is an agent that specifically and selectively binds andactivates the A₃AR. The A₃AR agonist has thus an IC₅₀ or a K_(i) that ispreferably at least 10, 15, 20, 25, 50, 75, 100, 150, 250 or at time atleast 500 times lower than the IC₅₀ or a K_(i) to any other adenosinereceptor.

The affinity of A₃AR agonists to the human A₃AR as well as its relativeaffinity to the other human adenosine receptors can be determined by anumber of assays, such as a binding assay. Examples of binding assaysinclude providing membranes or cells having the receptor and measuringthe ability of the A₃AR agonist to displace a bound radioactive agonist;utilizing cells that display the respective human adenosine receptor andmeasuring, in a functional assay, the ability of the A₃AR agonist toactivate or deactivate, as the case may be, downstream signaling eventssuch as the effect on adenylate cyclase measured through increase ordecrease of the cAMP level; etc. Clearly, if the administered level ofan A₃AR agonist is increased such that its blood level reaches a levelapproaching that of the K_(i) of the other adenosine receptors,activation of these receptors may occur following such administration,in addition to activation of the A₃R. An A₃AR agonist is thus preferablyadministered at a dose such that the blood level that will be attainedwill give rise to essentially only A₃R activation.

The characteristic of some adenosine A₃AR agonists and methods of theirpreparation are described in detail in, inter alia, U.S. Pat. No.5,688,774; U.S. Pat. No. 5,773,423; U.S. Pat. No. 5,573,772; U.S. Pat.No. 5,443,836; U.S. Pat. No. 6,048,865; WO 95/02604; WO 99/20284; WO99/06053; WO 97/27173 and WO/2006/031505 all of which are incorporatedherein by reference.

According to one embodiment of the invention, the A₃AR agonist is apurine derivative falling within the scope of the general formula (I):

wherein R₁ is C₁-C₁₀ alkyl, C₁-C₁₀ hydroxyalkyl, C₁-C₁₀ carboxyalkyl orC₁-C₁₀ cyanoalkyl or a group of the following general formula (II):

in which:

-   -   Y is oxygen, sulfur atom or CH₂;    -   X₁ is hydrogen, C₁-C₁₀ alkyl, R^(a)R^(b)NC(═O)— or HOR^(c)—,        wherein R^(a) and R^(b) may be the same or different and are        selected from hydrogen, C₁-C₁₀ alkyl, amino, C₁-C₁₀ haloalkyl,        C₁-C₁₀ aminoalkyl, C₁-C₁₀ BOC-aminoalkyl, and C₃-C₁₀ cycloalkyl        or are joined together to form a heterocyclic ring containing        two to five carbon atoms, and R^(c) is selected from C₁-C₁₀        alkyl, amino, C₁-C₁₀ haloalkyl, C₁-C₁₀ aminoalkyl, C₁-C₁₀        BOC-amino alkyl, and C₃-C₁₀ cycloalkyl;    -   X₂ is hydrogen, hydroxyl, C₁-C₁₀ alkylamino, C₁-C₁₀ alkylamido        or C₁-C₁₀ hydroxyalkyl;    -   X₃ and X₄ each independently are hydrogen, hydroxyl, amino,        amido, azido, halo, alkyl, alkoxy, carboxy, nitrilo, nitro,        trifluoro, aryl, alkaryl, thio, thioester, thioether, —OCOPh,        —OC(═S)OPh or both X₃ and X₄ are oxygen connected to >C═S to        form a 5-membered ring, or X₂ and X₃ form the ring of formula        (III):

where R′ and R″ are independently C₁-C₁₀ alkyl;

-   -   R₂ is selected from hydrogen, halo, C₁-C₁₀ alkylether, amino,        hydrazido, C₁-C₁₀ alkylamino, C₁-C₁₀ alkoxy, C₁-C₁₀ thioalkoxy,        pyridylthio, C₂-C₁₀ alkenyl; C₂-C₁₀ alkynyl, thio, and C₁-C₁₀        alkylthio; and    -   R₃ is a —NR₄R₅ group with R₄ being hydrogen or a group selected        from alkyl, substituted alkyl or aryl-NH—C(Z)—, with Z being O,        S, or NR^(a), and    -   when R₄ is hydrogen, R₅ being selected from R— and        S-1-phenylethyl, benzyl, phenylethyl or anilide groups, each        said groups being unsubstituted or substituted in one or more        positions with a substituent selected from C₁-C₁₀ alkyl, amino,        halo, C₁-C₁₀ haloalkyl, nitro, hydroxyl, acetoamido, C₁-C₁₀        alkoxy, and sulfonic acid or a salt thereof; or R₅ is        benzodioxanemethyl, fururyl, L-propylalanyl-aminobenzyl,        β-alanylamino-benzyl, T-BOC-β-alanylaminobenzyl, phenylamino,        carbamoyl, phenoxy or C₁-C₁₀ cycloalkyl; or R₅ is a group of the        following formula (IV):

-   -   or, when R₄ is alkyl, substituted alkyl, or aryl-NH—C(Z)—, then,        R₅ is selected from the group consisting of substituted or        unsubstituted heteroaryl-NR^(a)—C(Z)—, heteroaryl-C(Z)—,        alkaryl-NR^(a)—C(Z)—, alkaryl-C(Z)—, aryl-NR—C(Z)— and        aryl-C(Z)—;

or the A₃AR agonist is a xanthine-7-riboside derivative of the followinggeneral formula (V):

wherein:

-   -   X is O or S;    -   R₆ is R^(a)R^(b)NC(═O)— or HOR^(c)—, wherein    -   R^(a) and R^(b) may be the same or different and are selected        from hydrogen, C₁-C₁₀ alkyl, amino, C₁-C₁₀ haloalkyl, C₁-C₁₀        aminoalkyl, and C₃-C₁₀ cycloalkyl, or are joined together to        form a heterocyclic ring containing two to five carbon atoms;        and    -   R^(c) is selected from C₁-C₁₀ alkyl, amino, C₁-C₁₀ haloalkyl,        C₁-C₁₀ aminoalkyl, C₁-C₁₀ BOC-aminoalkyl and C₃-C₁₀ cycloalkyl;    -   R₇ and R₈ may be the same or different and are selected from        C₁-C₁₀ alkyl, C₁-C₁₀ cycloalkyl, R— or S-1-phenylethyl, an        unsubstituted benzyl or anilide group, and a phenylether of        benzyl group substituted in one or more positions with a        substituent selected from C₁-C₁₀ alkyl, amino, halo, C₁-C₁₀        haloalkyl, nitro, hydroxyl, acetamido, C₁-C₁₀ alkoxy, and        sulfonic acid;    -   R₉ is selected from the group consisting of halo, benzyl,        phenyl, C₃-C₁₀ cyclalkyl, and C₁-C₁₀ alkoxy;

or a suitable salt of the compound defined above.

In one embodiment, Y may form a fused bridge with either of thesubstituents X₁ or X₂. This embodiment is disclosed in WO 2006/031505,whose entire contents are incorporated by reference. In a furtherembodiment, such compounds may have the following general formula:

wherein X₁, R₂ and R₅ are as defined above.

According to another embodiment, the A3AR agonist is a nucleosidederivative of the general formula (VII):

wherein X₁, R₂ and R₅ are as defined above.

A non-limiting group of A₃AR agonists are theN⁶-benzyladenosine-5′-uronamide derivatives. Some preferredN⁶-benzyladenosine-5′-uronamide derivatives areN⁶-2-(4-aminophenyl)ethyladenosine (APNEA), N⁶-(4-amino-3-iodobenzyl)adenosine-5′-(N-methyluronamide)(AB-MECA),1-deoxy-1-{6-[({3-iodophenyl}methyl)amino]-9H-purine-9-yl}-N-methyl-β-D-ribofuranuronamide(IB-MECA) and 2-chloro-N⁶-(3-iodobenzyl)adenosine-5′-N-methlyuronamide(Cl-IB-MECA).

According to another embodiment, the A₃AR agonist isN⁶-benzyl-adenosine-5′-alkyluronamide-N¹-oxide orN⁶-benzyladenosine-5′-N-dialyl-uronamide-N¹oxide.

It is appreciated that the effective amount of the A₃AR agonist dependson a variety of factors including the affinity of the active agent toits corresponding receptor, its distribution profile within the body, avariety of pharmacological parameters such as half life in the body, onundesired side effects, if any, on factors such as weight, age, gender,treatment history, concomitant medications, and other parameters of thesubject to be treated, etc. The effective amount is typically tested inclinical studies having the aim of finding the effective dose range, themaximal tolerated dose and the optimal dose. The manner of conductingsuch clinical studies is well known to a person versed in the art ofclinical development.

An amount may also at times be deteimined based on amounts shown to beeffective in animals. It is well known that an amount of X mg/Kgadministered to rats can be converted to an equivalent amount in anotherspecies (notably humans) by the use of one of possible conversionsmethods well known in the art. Examples of conversion equations are asfollows:

Conversion I:

Body Surf. Species Body Wt. (Kg) Area (m²) Km Factor Mouse 0.2 0.00663.0 Rat 0.15 0.025 5.9 Human Child 20.0 0.80 25 Adult 70.0 1.60 37

Body Surface area dependent Dose conversion: Rat (150 g) to Man (70 Kg)is 1/7 the rat dose. This means that in the resent case 0.001-0.4 mg/Kgin rats equals to about 0.14-56 microgram/Kg in humans; assuming anaverage weight of 70 Kg, this would translate into an absolute dosage ofabout 0.01 to about 4 mg.

Conversion II:

The following conversion factors: Mouse=3, Rat=67. Multiply theconversion factor by the animal weight to go from mg/Kg to mg/m² forhuman dose equivalent.

Species Weight (Kg) BSA (m²) Human 70.00 1.710 Mouse 0.02 0.007 Rat 0.150.025 Dog 8.00 0.448

According to this equation the amounts equivalent to 0.001-0.4 mg/Kg inrats for humans are 0.16-64 μg/Kg; namely an absolute dose for a humanweighing about 70 Kg of about 0.011 to about 4.4 mg, similar to therange indicated in Conversion I.

Conversion III:

Another alternative for conversion is by setting the dose to yield thesame plasma level or AUC as that achieved following administration to ananimal.

An effective amount of an active agent may also be determined based onhuman PK studies. For example, human studies as described in US patentapplication, publication No. 20050101560 and by Fishman et al. [FishmanP. et al., Tolerability, pharmacokinetics, and concentration-dependenthemodynamic effects of oral CF101, an A3 adenosine receptor agonist, inhealthy young men Int J Clin Pharmacol Ther. 42:534-542, 2004, (CF101being a clinical grade, manufactured under cGMP guidelines, IB-MECA)]showed that the level of orally-administered IB-MECA decays in the humanplasma from its peak concentration with a half life of about 8-10 hours,as compared to a half life of only 1.5 hours in mice, in case ofmultiple daily administration, correction in the dosages foraccumulative effects needs to be made at times (a subsequent dose isadministered before the level of a previous one was decayed and thus,there is a build-up of plasma level over that which occurs in a singledose. On the basis of said human trials twice daily administrationappears to be a preferred administration regiment. However this does notrule out other administration regiments. Human studies conducted withCl-IB-MECA showed that the level of orally-administered Cl-IB-MECAdecays in the human plasma with a half life of about 12-14 hours fromits peak concentration. On the basis of this human data, theadministration regiment in human subjects may preferably be once ortwice daily although other regiments cannot be excluded.

In the context of the present invention, the pharmaceutical compositiontypically comprises a combination of an A3AR agonist with apharmaceutically acceptable carrier as well as other additives. Thecarrier may at times have the effect of the improving the delivery orpenetration of the active ingredient to the target tissue, for improvingthe stability of the drug, for slowing clearance rates, for impartingslow release properties, for reducing undesired side effects etc. Thecarrier may also be a substance that stabilizes the formulation (e.g. apreservative), for providing the formulation with an edible flavor, etc.Examples of carriers, stabilizers and adjuvants, are described, forexample, in E. W. Martin, REMINGTON'S PHARMACEUTICAL SCIENCES, MacK PubCo (June, 1990).

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used, is intended tobe in the nature of words of description rather than of limitation.Obviously, many modifications and variations of the present inventionare possible in light of the above teaching. It is therefore, to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described hereinafter.

DETAILED DESCRIPTION OF NON-LIMITING EXEMPLARY EMBODIMENTS Material &Methods Animals

Male Wistar rats (275-300 g) were fasted 12 h before the procedure. Therats were anaesthetized with Ketamin (45 mg/kg) and Xylazine (5 mg/kg).Laparatomy was performed via a sub costal, bilateral incision. The mainportal pedicle to the total liver was clamped for a period of 10minutes, during which, a 70% hepatectomy was performed. After 10 minutesof ischemia (in the course of the hepatectomy), blood flow was restoredby de-clamping.

Materials

The A₃AR agonist Cl-IB-MECA was synthesized for Can-Fite BioPharma byAlbany Molecular Research Inc, Albany, N.Y., USA. Cl-LB-MECA wasadministered at a concentration of 100 μg/kg starting at the end of theischemia, thrice daily for 48 h. Control rats did not receive Cl-IB-MECA

Methods

Rats underwent laparatomy and the main portal pedicle to the total liverwas clamped for a period of 10 minutes, during which a 70% hepatectomywas performed. After 10 minutes of ischemia blood flow was restored byde-clamping. Cl-IB-MECA (100 μg/Kg) was administered orally, TID,starting at the perfusion.

Results Hepatocyte Proliferation

With reference to FIG. 1, it may be seen CL-IB-MECA upregulatedhepatocyte proliferation of regenerated liver after partial hepatectomy,demonstrating a 45.1% level of regeneration in the treated group, ascompared to 30% in the control group.

Serum Levels of the Liver Enzymes ALT and AST

An increase in ALT and AST serum levels indicates liver damage. As canbe seen from FIGS. 2A and 2B, the ALT and AST levels were significantlydecreased in the CL-IB-MECA treated group as compared to the controlgroup at each of the three time points measured (2, 4 and 48 hours).

In a further assay, the effect of Cl-IB-MECA was compared to ratstreated with the vehicle only, or to rats that received no treatment andthe results are presented in FIG. 3. As shown, treatment with Cl-IB-MECAled to the reduction in serum levels of liver enzymes therebydemonstrating that Cl-IB-MECA treatment may protect liver against thedamage induced by the ischemia/reperfusion procedure.

Mitotic Index

Forty eight hours after partial hepatectomy the livers were collected,fixed with 10% buffered formalin and embedded in paraffin and 5 μm thicksections were subjected to Hematoxyline&Eosin staining. High powerfields (HPF, 400-fold magnification) were screened and cells undergoingmitosis were counted for each field. FIG. 4 presents an average of 50HPF counted. The results are presented as % of control. As shown,Cl-IB-MECA treatment significantly increased the mitotic index (P=0.035)in comparison to the vehicle-treated group.

Proliferating Cell Nuclear Antigen Expression

Proliferating cell nuclear antigen (PCNA) is a nuclear protein that isexpressed in the late G₁ and throughout the S-phase of the cell cycle.The amount of PCNA expression correlates with the degree of cellproliferation. Forty eight hours after partial hepatectomy the liverswere collected, fixed with 10% buffered formalin and embedded inparaffin and 5 μm thick sections were subjected to PCNA staining. Theamount of PCNA following treatment with vehicle only or with Cl-IB-MECAis shown in FIGS. 5A and 5B, respectively. As shown, Cl-IB-MECAtreatment significantly increased the number of PCNA positivehepatocytes (FIG. 5B).

Liver Regeneration

Livers were collected 24 and 48 hours after the partial hepatocytes andweight.

The growth of residual liver lobes was assessed using the followingequation:

${{Hepatic}\mspace{14mu} {regeneration}\mspace{14mu} {rate}\mspace{14mu} (\%)} = \begin{matrix}{C - \left( {A - B} \right)} \\{A \times 100}\end{matrix}$

where

A is the estimated total liver weight before hepatectomy (3.4% of arat's total weight),

B is the weight of liver resected during the hepatectomy, and

C is the weight of the regenerated liver at the end of the study.

A shown, Cl-IB-MECA treatment led to an increase in liver weight, and inother words, accelerated the rate of liver regeneration.

Thus, it was concluded that the A₃AR agonist Cl-IB-MECA is effective inregenerating the resected liver and preventing liver damage afterhepatectomy.

1-18. (canceled)
 19. A method of stimulating hepatocyte proliferation,comprising providing to a hepatocyte an agonist of the A₃ adenosinereceptor (A₃AR agonist) in an amount effective to stimulate hepatocyteproliferation.
 20. The method of claim 19, wherein the A₃AR agonist is apurine derivative falling within the scope of the general formula (I):

wherein R₁ is C₁-C₁₀ alkyl, C₁-C₁₀ hydroxyalkyl, C₁-C₁₀ carboxyalkyl orC₁-C₁₀ cyanoalkyl or a group of the following general formula (II):

in which: Y is oxygen, sulfur atom or CH₂; X₁ is hydrogen, C₁-C₁₀ alkyl,R^(a)R^(b)NC(═O)— or HOR^(c)—, wherein R^(a) and R^(b) may be the sameor different and are selected from hydrogen, C₁-C₁₀ alkyl, amino, C₁-C₁₀haloalkyl, C₁-C₁₀ aminoalkyl, C₁-C₁₀ BOC-aminoalkyl, and C₃-C₁₀cycloalkyl or are joined together to form a heterocyclic ring containingtwo to five carbon atoms, and R^(c) is selected from C₁-C₁₀ alkyl,amino, C₁-C₁₀ haloalkyl, C₁-C₁₀ aminoalkyl, C₁-C₁₀ BOC-aminoalkyl, andC₃-C₁₀ cycloalkyl; X₂ is hydrogen, hydroxyl, C₁-C₁₀ alkylamino, C₁-C₁₀alkylamido or C₁-C₁₀ hydroxyalkyl; X₃ and X₄ each independently arehydrogen, hydroxyl, amino, amido, azido, halo, alkyl, alkoxy, carboxy,nitrilo, nitro, trifluoro, aryl, alkaryl, thio, thioester, thioether,—OCOPh, —OC(═S)OPh or both X₃ and X₄ are oxygen connected to >C═S toform a 5-membered ring, or X₂ and X₃ form the ring of formula (III):

where R′ and R″ are independently C₁-C₁₀ alkyl; R₂ is selected fromhydrogen, halo, C₁-C₁₀ alkylether, amino, hydrazido, C₁-C₁₀ alkylamino,C₁-C₁₀ alkoxy, C₁-C₁₀ thioalkoxy, pyridylthio, C₂-C₁₀ alkenyl; C₂-C₁₀alkynyl, thio, and C₁-C₁₀ alkylthio; and R₃ is a —NR₄R₅ group with R₄being hydrogen or a group selected from alkyl, substituted alkyl oraryl-NH—C(Z)—, with Z being O, S, or NR^(a), and when R₄ is hydrogen, R₅being selected from R- and S-1-phenylethyl, benzyl, phenylethyl oranilide groups, each said groups being unsubstituted or substituted inone or more positions with a substituent selected from C₁-C₁₀ alkyl,amino, halo, C₁-C₁₀ haloalkyl, nitro, hydroxyl, acetoamido, C₁-C₁₀alkoxy, and sulfonic acid or a salt thereof; or R₅ isbenzodioxanemethyl, fururyl, L-propylalanyl-aminobenzyl,β-alanylamino-benzyl, T-BOC-β-alanylaminobenzyl, phenylamino, carbamoyl,phenoxy or C₁-C₁₀ cycloalkyl; or R₅ is a group of the following formula(IV):

or, when R₄ is alkyl, substituted alkyl, or aryl-NH—C(Z)—, then, R₅ isselected from the group consisting of substituted or unsubstitutedheteroaryl-NR^(a)—C(Z)—, heteroaryl-C(Z)—, alkaryl-NR^(a)—C(Z)—,alkaryl-C(Z)—, aryl-NR—C(Z)— and aryl-C(Z)—; or the A₃AR agonist is axanthine-7-riboside derivative of the following general formula (V):

wherein: X is O or S; R₆ is R^(a)R^(b)NC(═O)— or HOR^(c)—, wherein R^(a)and R^(b) may be the same or different and are selected from hydrogen,C₁-C₁₀ alkyl, amino, C₁-C₁₀ haloalkyl, C₁-C₁₀ aminoalkyl, and C₃-C₁₀cycloalkyl, or are joined together to form a heterocyclic ringcontaining two to five carbon atoms; and R^(c) is selected from C₁-C₁₀alkyl, amino, C₁-C₁₀ haloalkyl, C₁-C₁₀ aminoalkyl, C₁-C₁₀ BOC-aminoalkyland C₃-C₁₀ cycloalkyl; R₇ and R₈ may be the same or different and areselected from C₁-C₁₀ alkyl, C₁-C₁₀ cycloalkyl, R- or S-1-phenylethyl, anunsubstituted benzyl or anilide group, and a phenylether of benzyl groupsubstituted in one or more positions with a substituent selected fromC₁-C₁₀ alkyl, amino, halo, C₁-C₁₀ haloalkyl, nitro, hydroxyl, acetamido,C₁-C₁₀ alkoxy, and sulfonic acid; R₉ is selected from the groupconsisting of halo, benzyl, phenyl, C₃-C₁₀ cyclalkyl, and C₁-C₁₀ alkoxy;or a suitable salt of the compound defined above.
 21. The method ofclaim 20, wherein the A₃AR agonist is a N⁶-benzyladenosine-5′-uronamidederivative.
 22. The method of claim 21, wherein the A₃AR agonist isselected from the group consisting of N⁶-2-(4-aminophenyl)ethyladenosine(APNEA),N⁶-(4-amino-3-iodobenzyl)adenosine-5′-(N-methyluronamide)(AB-MECA),1-deoxy-1-{6-[({3-iodophenyl}methyl)amino]-9H-purine-9-yl}-N-methyl-β-D-ribofuranuronamide(IB-MECA) and 2-chloro-N⁶-(3-iodobenzyl)adenosine-5′-N-methlyuronamide(Cl-IB-MECA).
 23. The method of claim 22, wherein the A₃AR agonist isIB-MECA or Cl-IB-MECA.
 24. The method of claim 23, wherein the A₃ARagonist is Cl-IB-MECA.
 25. The method of claim 19, for inducinghepatocyte proliferation in liver following liver damage ordisease-induced damage.
 26. The method of claim 23, for inducinghepatocyte proliferation in liver following liver damage ordisease-induced damage.
 27. The method of claim 25, wherein the liverdamage or disease-induced damage is a result of hepatectomy, cirrhosisof the liver, hepatic malignancies, exposure to alcohol, hepatotoxicdrugs and combinations thereof, infectious agents, the side effects ofgene therapy, exposure to anti-tuberculosis agents and chemotherapeuticagents, or acetaminophen (APAP) overdoses.
 28. The method of claim 26,wherein the liver damage or disease-induced damage is a result ofhepatectomy, cirrhosis of the liver, hepatic malignancies, exposure toalcohol, hepatotoxic drugs and combinations thereof, infectious agents,the side effects of gene therapy, exposure to anti-tuberculosis agentsand chemotherapeutic agents, or acetaminophen (APAP) overdoses.
 29. AnA₃AR agonist for use in a method for stimulating hepatocyteproliferation.
 30. An A₃AR agonist for use in a method for treating apatient having liver damage due to hepatectomy.
 31. The A₃AR agonist ofclaim 29 selected from the group consisting ofN⁶-2-(4-aminophenyl)ethyladenosine (APNEA), N⁶-(4-amino-3-iodobenzyl)adenosine-5′-(N-methyluronamide) (AB-MECA),1-deoxy-1-{6-[({3-iodophenyl}methyl)amino]-9H-purine-9-yl}-N-methyl-β-D-ribofuranuronamide(IB-MECA) and 2-chloro-N⁶-(3-iodobenzyl)adenosine-5′-N-methlyuronamide(Cl-IB-MECA).
 32. The A₃AR agonist of claim 31, being IB-MECA orCl-IB-MECA.
 33. The A₃AR agonist of claim 32, being Cl-IB-MECA.